Calcium, as a second messenger, plays a major regulatory role in a wide variety of cellular processes ranging from the control of gene expression, protein phosphorylation, actin-myosin interactions, tublin polymerization and depolymerization, to membrane vesicle fusion. Calcium is also involved in biochemical and cellular pathology. Cytoplasmic calcium levels are governed by a homeostatic balance between influx and clearance mechanisms. A calcium signal transient temporarily disturbs this homeostatic balance; calcium clearance mechanisms restore the calcium levels. This proposal examines the spatial and temporal distribution of cytoplasmic calcium in neuronal cell bodies following entry through voltage-gated channels. A stationary slit confocal microscope will be used to measure calcium indicator dye fluorescence along a narrow plane of focus through the cell body of identified cultured neurons from snail and from mouse dorsal root ganglia. This approach eliminates the contamination from membrane regions that is an inherent pitfall of standard epifluorescent microscopy. Fluorescence will be projected onto a linear photodiode array to measure calcium concentrations at 10 millisecond time resolution at different depths within the neuron. Voltage-clamp pulses will induce a quantifiable calcium influx. Computer modelling of the calcium transient will estimate the activity of the calcium clearance, buffering, and sequestration mechanisms. Calcium sequestering and releasing organelles will be observable as irregularities in the calcium transient; those organelles will be identified with organelle specific fluorescent labels and with transmission electron microscopy. The local distribution of voltage-gated calcium channels and calcium clearance activities will be measured. The role of calcium clearance mechanisms in regulating the magnitude and timecourse of the calcium transient will be determined. The regulation of the calcium transient by neuronal growth state, electrical activity, and various second messengers will also be examined. The studies in this proposal will provide direct measurements of the diffusion of calcium within single neurons. Taken together, the results will address the regulation and the location of calcium clearance mechanisms, the location of calcium sequestration or releasing organelles, and the role of calcium clearance mechanisms in governing cytoplasmic calcium transients in the neuronal cell body. They will also provide direct evidence for or against local calcium cycling at the plasma membrane. Thus, these studies will provide insight into, and stimulate further studies of, the regulation of calcium transients in other regions of the neuron such as pre- and post-synaptic structures.